Hydrogen alleviates hypoxic–ischaemic brain damage in neonatal rats by inhibiting injury of brain pericytes

Abstract Hypoxia‐ischaemia (HI) can induce the death of cerebrovascular constituent cells through oxidative stress. Hydrogen is a powerful antioxidant which can activate the antioxidant system. A hypoxia‐ischaemia brain damage (HIBD) model was established in 7‐day‐old SD rats. Rats were treated with different doses of hydrogen‐rich water (HRW), and brain pericyte oxidative stress damage, cerebrovascular function and brain tissue damage were assessed. Meanwhile, in vitro‐cultured pericytes were subjected to oxygen–glucose deprivation and treated with different concentrations of HRW. Oxidative injury was measured and the molecular mechanism of how HRW alleviated oxidative injury of pericytes was also examined. The results showed that HRW significantly attenuated HI‐induced oxidative stress in the brain pericytes of neonatal rats, partly through the Nrf2‐HO‐1 pathway, further improving cerebrovascular function and reducing brain injury and dysfunction. Furthermore, HRW is superior to a single‐cell death inhibitor for apoptosis, ferroptosis, parthanatos, necroptosis and autophagy and can better inhibit HI‐induced pericyte death. The liver and kidney functions of rats were not affected by present used HRW dose. This study elucidates the role and mechanism of hydrogen in treating HIBD from the perspective of pericytes, providing new theoretical evidence and mechanistic references for the clinical application of hydrogen in neonatal HIE.

cerebral vascular dysfunction caused by hypoxia ischaemia (HI) is an important factor leading to the occurrence and development of brain injury. 4ricytes are the main cells that make up cerebral blood vessels.
They are embedded in the basement membrane of cerebral microvascular endothelial cells and play an important role in maintaining the blood-brain barrier (BBB), regulating cerebral blood flow (CBF) and modulating immune cell entry into the central nervous system. 5erefore, pericytes play a key role in the occurrence and development of central nervous system diseases.Under normal physiological conditions, increased neuronal excitability or neurotransmitter release can cause pericytes to relax, leading to capillary dilation.
When cerebral ischaemia occurs, many pericytes die, leading to irreversible contraction of capillaries and severe cerebrovascular dysfunction. 6Another study found that death and sustained abnormal contraction of pericytes during ischaemia led to a reduction in capillary diameter and the arrangement of red blood cells appeared intermittent, affecting blood flow perfusion. 7NO synthase inhibitors and oxygen-free radical scavengers can reduce pericyte death, thus relieving sustained abnormal contraction of pericytes and restoring microcirculation. 8,9Pericyte death inhibition can be expected to become an important way to alleviate cerebrovascular dysfunction after HI.
Cerebral vessels are highly sensitive to oxidative stress.HI induces NO synthase activation in vascular endothelial cells, leading to NO production.Superoxide anions (O2 − ) are also produced during vascular damage.O2 − reacts with NO to form the peroxynitrite anion (ONOO − ), which is a strong inducer of DNA damage that can cause oxidative stress in vascular cells. 10Because the number of dead pericytes after HI is much higher than vascular endothelial cells, the study of pericyte death is particularly important. 4drogen (H 2 ) is a powerful antioxidant that activates the body's antioxidant system and effectively reduces O2 − generation. 11Therefore, we hypothesize that H 2 can alleviate HI-induced oxidative damage in pericytes caused by HI, maintain pericyte survival and improve cerebrovascular function.In 2007, Ohta first investigated the effect of H 2 on brain injury and found that H 2 can selectively clear cytotoxic free radicals OH and ONOO − , exhibiting a protective effect on acute brain ischaemia/reperfusion injury. 12Subsequently, H 2 role in neurological diseases was widely promoted.To date, increasing evidence has shown protective effects of H 2 against various neurological diseases. 13Moreover, at least 10 clinical trials have been conducted on the use of H 2 in nervous system diseases, showing that H 2 is a promising therapeutic medical gas for brain disorders. 13However, these studies focus mainly on adult neurological diseases, and there are relatively few studies on neonatal brain injury.Moreover, these studies mainly involve neurons, microglia and astrocytes, with almost no reports on pericytes.As pericytes role in regulating neurovascular coupling and neurological diseases becomes increasingly important, we plan to focus on exploring the improvement of pericyte biological behaviour through H 2 therapy.This study established a hypoxic-ischaemic brain damage (HIBD) model of neonatal rats and an in vitro oxygen and glucose deprivation (OGD) model of pericytes to elucidate the effects and mechanisms of H 2 on pericyte death, cerebrovascular function and brain function through in vivo and in vitro experiments to provide new theoretical evidence and mechanism references for the clinical application of H 2 in HIE.

| Establishment of the HIBD model in neonatal rats
All animal experiments in this study were approved by the Animal Ethics Review Committee of Sichuan University (Animal Ethics Review Pass No. WCSUH21-2018-034); 7-day-old SD rats (16-21 g, male and female) were purchased from Sichuan Dasuo Laboratory Animal Co., Ltd., China.The HIBD model was established using the Rice-Vannucci method. 14Briefly, experimental rats were placed on a small-animal special anaesthesia machine (Matrix, USA) and inhaled isoflurane.The right common carotid artery was severed with an electric coagulation knife (Aesculap, Germany).After surgical suture, the rats were recovered for 1 h, then placed in a low oxygen chamber (temperature, 37°C; 92% N 2 and 8% O 2 ) and the mixture gas was ventilated at 0.5-1 L/min for 2 h.

| Hydrogen-rich water (HRW) treatment
HRW (concentration: 1000 μmol/L) was purchased from Beijing Fuhydrogen Source Technology Co., China.The 7-day-old SD rats were randomly assigned to seven groups: normal control group: no treatment; sham group: the right common carotid artery was isolated without electrocoagulation or hypoxic ventilation; HIBD group: the right common carotid artery was isolated by electrocoagulation ionization and hypoxic ventilation; HI + HRW treatment group: after successful HIBD moulding, rats were injected intraperitoneally with HRW.The first injection was conducted immediately after moulding and the same dose of HRW was injected 24, 48 and 72 h after moulding.This group was further divided into three subgroups according to the different doses of HRW used: (1) HI + HRW-5 (5 mL/ kg), (2) HI + HRW-10 (10 mL/kg) and (3) HI + HRW-15 (15 mL/kg); HI + solvent group: an equal amount of distilled water was injected intraperitoneally at the time points corresponding to HRW administration.Rats in each group were killed at different time points for further examination.

| Propidium iodide (PI) staining
PI (5 mg/kg; Sigma, USA) was injected into the tail vein of rats.After circulation for 1 h, rats were killed, their brains were removed and frozen sections were prepared.The frozen sections were baked at 37°C for 1 h, washed with PBS for 5 min and then incubated with β-PDGFR antibody (1:100; Abcam, United Kingdom) or NeuN antibody (1:200; Abcam, United Kingdom).Finally, the sections were stained with DAPI and observed under the fluorescence microscope (Olympus, FV1000, Japan).Triple positive staining of DAPI/PI/β-PDGFR was calculated to examine pericyte death, and triple positive staining of DAPI/PI/NeuN was calculated to examine neuronal death.

| SOD activity
SOD activity in brain tissues was detected using an SOD detection kit (Shanghai Biyun Biotechnology Company, China).

| Tissue immunofluorescence staining
Rat brains were dissected and embedded in an optimal cutting temperature (OCT) compound (Tissue-Tek, Torrance, USA), and then cryosectioned to 20μm thickness and fixed in ice-cold acetone.Sections were blocked with 5% normal swine serum (Vector Laboratories, Burlingame, USA) for 60 min and incubated in primary antibody overnight at 4°C.The sections were washed in PBS and incubated with Cy3-or Cy5-conjugated secondary antibodies.Primary antibodies used are β-PDGFR (1:100; Novus, USA) and ROS (1:100; Beijing Boosen Biological Company, China).Images were scanned using a digital slice scanner (3DHISTECH, Hungary) and analysed using ImageJ software.

| Detection of brain vessel diameter and pericyte coverage
FITC-labelled tomato lectin (Shanghai Maokang Biotechnology, China, 100 μg per rat) was injected into rats by tail vein.Rats were killed 15 min later, and the brain was prepared in frozen sections for immunofluorescence staining with β-PDGFR antibody (1:100; Abcam, United Kingdom).The images were scanned using a digital slice scanner, the scanned images were superimposed and the vessels could be observed clearly.ImageJ software was used to select brain microvessels with a diameter <10 μm as the analysis object.
The diameter was measured every 2 μm along the long axis of microvessels with a total length of 100 μm, and the average diameter was calculated.The number of β-PDGFR-positive pericytes covered on the microvascular per unit length (100 μm) was calculated to reflect pericyte coverage.

| Evans blue (EB) staining
A 2% EB solution (2 mL/kg; Sigma) was injected into the rats through the tail vein.After circulation for 2 h, rats were killed and their brain tissue was homogenized in trichloroacetic acid (Shanghai Maclin Biochemical Technology company, China), and then centrifuged at 4°C, 3000 g for 20 min.The supernatant was mixed with 100% ethanol in a 1:3 ratio to prepare a mixed solvent.The mixed solvent (200 μL) was placed in a 96well plate, and the OD value was measured at 562 nm using a microplate reader (Molecular Devices, USA); then, EB content can be calculated from the standard curve.

| Laser speckle imaging
The rats were anesthetized with isoflurane and their head skin was cut open to fully expose the brain.The laser beam from the RFLSI III laser speckle imaging system (Shenzhen Rayward Life Technology Company, China) was focused on the cerebral cortex 2 mm from the front fontanels and 3 mm from the middle line.Blood flow was continuously detected for 60 s and the mean CBF in the left and right cerebral hemispheres was analysed.

| Haematoxylin and eosin (H&E) staining
Brain tissue was fixed with 4% paraformaldehyde, dehydrated with alcohol, soaked in xylene, embedded in paraffin, then cut into 5 μm slices and dried, dewaxed in xylene, treated with alcohol and stained with haematoxylin-eosin.An optical microscope (Olympus) was used to observe the pathological changes in the sections.

| Triphenyltetrazolium chloride monohydrate (TTC) staining
The rat cerebral infarction volume was assessed using TTC staining (Sigma, USA).Each brain section was cut into four pieces and the thickness of each slice was 2 mm.The slices were then placed in a 2% TTC solution, incubated at 37°C for 20 min, the TTC solution was removed, fixed with 4% paraformaldehyde and photographed.
Image-Pro Plus software (version 6.0) was used to calculate the infarct volume.The infarct volume/brain volume ratio was used to determine the percentage of infarcts.

| Neurofunctional score
The severity of neurological damage was evaluated using the Zea-Longa score 15

| Morris water maze (MWM) test
The MWM test was conducted to evaluate the neurocognitive and motor coordination ability of rats 29 days after birth. 16For the first 5 days (P29-P33), each rat was trained to swim in the four quadrants of the circular pool to detect its position navigation ability.The average escape latency time in the four quadrants was calculated and recorded as a daily final score, representing the ability to acquire spatial information.At P34, the place navigation training was conducted.The platform was removed, and the rats were released at the farthest point from the platform.The rats were allowed to swim for 60 s, and platform-crossing times were recorded.

| Cell culture
Rat cerebral pericytes were isolated according to the established protocols. 17 and the grey matter was cut into 1 mm pieces in an ice-cold Dulbecco's modified Eagle's medium (DMEM, Gibco, Rockville, USA).Homogenates were digested with collagenase type II (1 mg/ mL; Sigma, St. Louis, USA) and DNase I (37.5 mg/mL; Sigma) in DMEM containing penicillin (100 U/mL) and streptomycin (100 mg/ mL) at 37°C for 1.5 h with agitation.Neurons and glial cells were removed by centrifugation in 20% bovine serum albumin (BSA)-DMEM (1000 × g for 20 min).Microvessels obtained from the pellets were further digested with collagenase type II (1 mg/mL) and DNase I (16.7 mg/mL) in DMEM at 37°C for 45 min with agitation.Microvessel endothelial cell clusters were separated using 33% continuous Percoll (GE Healthcare, Uppsala, Sweden) gradient centrifugation (1000 × g for 10 min).Endothelial cell clusters were pipetted and filtered through 70μm nylon mesh.Cell pellets were washed twice with DMEM (first at 1000 × g for 8 min, then at 700 × g for 5 min) and placed in uncoated culture flasks in DMEM supplemented with 10% FBS, L-glutamine (2 mM), glucose (4.5 g/L), penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37°C with a humidified atmosphere with 5% CO 2 .Cells were allowed to adhere for 4-5 h, and then non-adherent cells were removed.After 14 days in culture, rat pericytes overgrew brain endothelial cells and reached 80%-90% confluence.Cells were used in passages 2-3 with a purity of >95% determined with NG2 and PDGFRβ immunostaining.

| OGD of cultured pericytes
Glucose deprivation (GD) was achieved by replacing the pericyte growth medium with glucose-free DMEM (Gibco).Cells in GD medium were then transferred to a humidified anaerobic chamber containing 94% N 2 /5% CO 2 /1% O 2 at 37°C.The OGD was terminated by replacing the GD medium with a normal pericyte growth medium, and the cells were returned to a normoxic incubator.Cells grown under normal conditions were used as controls.

| Cell death inhibitor treatment for cultured pericytes
To compare the effect of selected inhibitors from different cell death models with HRW, immediately after OGD treatment, pericytes were treated with 100 μM Z-VAD.FMK (Abcam, Cambridge, MA, USA), 1 μM Fer-1 (Sigma), 20 μM DPQ (Abcam), 30 μM Nec-1 (Abcam), 10 mM 3-MA (Sigma) or 10 μmol/L HRW for 24 h.The control group was treated with a vehicle.The drug dosing schedule was determined based on the results of previous reports. 18,19

| Cell survival assay
Cell survival assays were performed using a Cell Counting Kit-8 (CCK-8 kit, Dojindo Co., Japan) according to the manufacturer's instructions.The luminescence was recorded using a Varioskan flash microplate reader (Thermo Fisher Scientific, Waltham, USA).

| Statistical analysis
The number of positive cells, cerebrovascular diameter and cerebrovascular peripheral cell coverage in the immunofluorescence images were calculated using ImageJ software.SPSS 22.0 software was used to process the experimental data and the data were expressed by mean ± SEM.Comparisons between two groups were performed using a t-test and comparisons between multiple groups were performed using a one-way analysis of variance (ANOVA).Statistical significance was set at p < 0.05.Statistical histograms were plotted using GraphPad Prism 7.0.

| Effects of HRW on HI-induced oxidative injury in pericytes in vivo and in vitro
H&E staining showed that after HI, the right cerebral cortex of the rats was loose, cells were disorderly arranged and some cells were severely necrotic.After HRW treatment, the pathological damage and infarct area in the brain tissue were significantly reduced.HRW-10 was more effective than HRW-5, and there was no significant difference between the HRW-15 and HRW-10 groups (Figure 1A).Therefore, we chose HRW-10 as the treatment dose in subsequent experiments.Furthermore, there was no significant difference between the HI and HI + solvent groups (Figure 1A); we did not include the HI + solvent group in subsequent experiments.Immunostaining showed that, after HI, the number of DAPI/PI/β-PDGFR-positive cells in brain tissue increased, indicating obvious death of pericytes (Figure 1B).Meanwhile, the number of β-PDGFR/ROS-positive cells increased (Figure 1C), and the number of β-PDGFR/SOD-positive cells decreased (Figure 1D study.Immunofluorescence staining was used to detect specific markers (α-SMA and β-PDGFR) in cultured pericytes.It showed that most cells expressed α-SMA or β-PDGFR, while the endothelial cell marker CD31 and the astrocyte marker GFAP were negatively stained (Figure 2A), indicating a high purity of cultured pericytes.
After OGD, the survival of cultured pericytes decreased and their death increased; treatment of pericytes with HRW-containing culture medium resulted in a significant increase in pericyte survival and a decrease in cell death, with 10 μmol/L HRW as the most effective and safe dose (Figure 2B).The SOD activity of pericytes increased, while ROS and 8-OHdG concentrations in pericytes decreased with HRW treatment after OGD (Figure 2C).Furthermore, HRW was superior to a single-cell death inhibitor and better inhibited HI-induced pericyte death (Figure 3).

| Effects of HRW on cerebrovascular function in HIBD rats
Quantitative analyses of lectin staining on confocal laser scanning microscopy stacked images assessing the diameter of brain blood vessels showed that HI-induced capillary constriction, with the diameter of brain vessels being significantly smaller in rats after HI than in sham controls, while HRW broadened the brain vessels (Figure 4A).Using dual immunostaining for β-PDGFR and lectin, we showed a loss of pericyte coverage in HI brain compared to sham controls, while HRW increased the pericyte coverage (Figure 4B).Furthermore, the mean surface CBF on the ipsilateral side of HI rats was significantly lower than in sham controls after HI, and HRW significantly increased the ipsilateral CBF of HI rats (Figure 4C).Furthermore, extravasation of EB dye in the ipsilateral hemisphere was greater in HI rats than in sham controls, while HRW decreased BBB breakdown in the ipsilateral hemisphere of HI rats (Figure 4D).
Since HRW effectively restored HI-damaged brain vessel function, we further examined its neuroprotective effects on brain function in a rat model.Triple positive staining of DAPI/ PI/NeuN showed that HI induced neuronal death, while HRW significantly attenuated it (Figure 5A).TTC staining showed that HI caused severe infarcts in rat brains, while HRW decreased the infarct volume from approximately 42%-23% at 7 days after HI (Figure 5B).We determined the long-term effects of HRW on neurological outcomes.NSS evaluation 21 days after HI showed that NSS was significantly higher in the HI group than in the sham group and decreased after HRW treatment (Figure 5C).To further verify the long-term neurological protection of HRW in rats after HI, the MWM test was performed to evaluate spatial learning and memory 34 days after HI.The escape latency of all groups showed a downward trend in the MWM test (Figure 5D).
Compared to the HI group, rats in the HRW group took significantly less time to find the underwater platform in the second quadrant on days 4 and 5 and crossed the former platform more times.

| Liver and kidney function after HRW treatment
The liver and kidney functions (AST, ALT and BUN) of the Sham, HI and HRW groups were within the normal range (Figure 6A).Pathological changes in the liver and kidneys of the three groups were observed by H&E staining.The structures of the hepatic lobule and sinusoids were clear, the structure of the hepatic cell cord was orderly arranged and the hepatic cells were radially arranged.It also showed a normal glomerular structure without obvious proliferation of mesangial cells and mesangial matrix, no obvious inflammatory cells entering the renal interstitium and no abnormalities in the renal tubule structure (Figure 6B).

| HRW upregulated NRF2 and heme oxygenase 1 in OGD pericytes
H 2 has been known to decrease ROS through its radical scavenger and mitochondria rectifier activity. 12,20Furthermore, H 2 might evoke Nrf2-HO-1 pathway to activate antioxidant defence system, 21,22 so we detected the expression of Nrf2 and HO-1 in OGD pericytes.
Western blot showed that there was little expression of Nrf2 and HO-1 in control pericytes.After OGD, the expression of these genes was not significantly affected, while HRW significantly upregulated their expression (Figure 7).

| DISCUSS ION
This study simulated the pathological damage of HIE using ani- Mechanistically, H 2 can decrease oxidative stress in pericytes after HI, in addition to a canonical mechanism, perhaps by inducing the Nrf2-HO-1 pathway.
Previous reports have shown that H 2 therapy mechanisms for brain injury include mainly antioxidant, anti-inflammatory and anti-apoptotic effects. 13These mechanisms are interconnected in a complex manner.Oxidative stress affects the expression of numerous genes, such as those related to cell apoptosis and inflammatory response; Therefore, the antioxidant effect of H2 may be its most fundamental property. 23To date, there are two main hypotheses on the antioxidant mechanism of H 2 .The widely accepted hypothesis is that H 2 reacts directly with OH and ONOO − , which is the traditional 'scavenger theory'. 12Another view is that H 2 inhibits ROS production by acting as a rectifier for mitochondrial electron flow. 20In addition to its properties as a free radical scavenger and rectifier of the mitochondrial respiratory chain, H 2 treatment might induce adaptive responses to oxidative stress by evoking the Nrf2 antioxidant defence system. 21,22f2 a positive regulator of the human antioxidant response element, modulating the expression of hundreds of genes, most of which are antioxidant genes, including HO-1 and SOD. 24It has been reported that a relevant source of ROS stems from mitochondrial complex I inhibition; HO-1 upregulation can protect against complex I inhibitors and decrease ROS production. 25,26erefore, HO-1 is considered an important anti-oxygen stress enzyme that plays a role in many types of tissue injury.Based on this, we examined Nrf2 and HO-1 levels using western blotting.It showed that HRW treatment significantly increased Nrf2 and HO-1 expression in pericytes, suggesting that, besides the canonical mechanism, the Nrf2-HO-1 pathway is another HRW mechanism to modulate ROS production and subsequent oxidative stress injury.HI can induce various forms of cell death. 27To compare the effect of different death inhibitors and HRW on pericyte death after HI, we tested OGD-induced cell death after treatment with inhibitors of different death modes.The results showed that the death patterns of brain pericytes induced by HI include apoptosis, parthanatosis and ferroptosis; however, by inhibiting a single specific death mode, pericytes death cannot be fully suppressed.Previously, oxidative stress was shown to be the cause of multiple modes of cell death. 28When subjected to various harmful stimuli, excessive ROS and reactive nitrogen species are produced in cells. 29Excess ROS and reactive nitrogen species can lead to lipid peroxidation and oxidative damage to proteins and DNA.Membrane lipid peroxidation not only leads to changes in membrane function but also to structural damage, ultimately leading to cell rupture.Membrane lipid peroxidation is involved in various types of programmed cell death, such as ferroptosis, 30 necroptosis, 31 parthanatos, 32 autophagy, 33 etc.Oxidative DNA damage is closely related to the occurrence of apoptosis and parthanatos. 34,35Given that HI-induced activation of apoptosis, parthanatosis and ferroptosis is closely related to the earlier response to oxidative stress in cells, reducing oxidative stress of pericytes is an effective strategy to inhibit pericyte damage.In this study, we found that HRW was superior to a single-cell death inhibitor and better inhibited HI-induced pericyte death, confirming this conjecture.
Hydrogen has several advantages as a neuroprotective gas.It can cross the BBB, penetrate the cell membrane and diffuse into the cytosol and organelles. 13Furthermore, repeated administration of H 2 does not cause tolerance. 36There are multiple routes of H 2 administration, including inhalation of H 2 gas, drinking HRW, injection of H 2 -rich saline (HRS), HRW bathing, intake of a solid H 2 carrier (coral calcium hydride) and H 2 -producing precursors, as well as functional micro/nanomaterials for targeted H 2 delivery. 13ch method of administration has its advantages and disadvantages. 37,38As 7-day-old newborn rats cannot drink water spontaneously, in this experiment, HRW is administered intraperitoneally.
Considering that babies born with severe asphyxia usually require assisted ventilation, inhalation of H 2 gas could be a direct and effective way of H 2 administration for babies with neonatal HIE. 39though many studies have shown that H 2 has no toxic effects, adverse events, such as diarrhoea and heartburn have been reported in individual cases. 13Fortunately, this study showed that the liver and kidney functions of rats were unaffected when using the present dose of HRW.
Several studies have shown the neuroprotective effect of H 2 on neonatal HIBD.Intraperitoneal injection of HRS significantly promotes M2 microglia polarization and suppresses neuroinflammation, further restoring behavioural deficits in a neonatal mouse model of HI. 40 Similar results have been obtained from HI models in neonatal rats and piglets. 41,42In a rat model of neonatal HIBD, H 2 inhalation has been shown to inhibit neuronal loss and astrocyte activation, further reducing the infarct size of the brain. 41other study in a 5-day neonatal hypoxia/ischaemia piglet model showed that H 2 ventilation combined with mild hypothermia improves the neurological score and improves the motor function of piglets. 42A recent study showed that H 2 combined with therapeutic hypothermia ameliorated seizure burden after HI insult in newborn piglets. 43These studies mainly involve the effects of H 2 on neurons, microglia and astrocytes.There have also been studies attempting to explore H 2 gas role from the perspective of regulating neurovascular reactivity, 44,45 but no in-depth research has been conducted on its mechanism.In recent years, neurovascular coupling role in physiological function and pathological damage to the nervous system has been increasingly valued. 46Recent studies have found that pericytes are a key factor in the neurovascular coupling system, 47 prompting researchers to further explore pericytes role in neurological diseases.Our study elucidates the role and mechanism of H 2 in treating HIBD from the perspective of pericytes, providing new theoretical evidence and mechanism references for the clinical application of ; 0 point indicated completely normal behaviour without any neurological deficit; 1 point indicated opposite forelimb flexion, mild neurological deficit; 2 points indicated crawling opposite rotation, moderate neurological impairment; 3 points indicated standing or crawling opposite dumping, severe neurological impairment; and 4 points indicated no autonomous activity with consciousness impairment.The scoring process in this experiment was performed by three staff in double-blind.
mal and cell models and clarified that HRW can inhibit pericyte injury and improve cerebrovascular and brain function after HI.F I G U R E 3 HRW is superior to a single-cell death inhibitor and can better inhibit HI-induced pericyte death.Data are represented as mean ± SEM (n = 6).Ctr, without OGD treatment; V, vehicle control; VAD, apoptosis inhibitor; Fer, ferroptosis inhibitor; DPQ, parthanatosis inhibition; Nec-1, necroptosis inhibitor; 3-MA, autophagy inhibitor.*p < 0.05.

F I G U R E 5
Effects of HRW on neurological function in HIBD rats.(A) Triple positive staining of DAPI/PI/NeuN to show neuronal death.Data are represented as mean ± SEM (n = 3).Scale bar = 20 μm.(B) Brain infarcts are detected using TTC staining.Data are represented as mean ± SEM (n = 3).(C) Evaluation of NSS.The experiment is carried out three times, n = 5 rats per group.Data provided as mean ± SEM. (D) Spatial learning and memory abilities of rats detected using the MWM.Data are represented as mean ± SEM (n = 9).HI + HRW, HI + HRW-10 (10 mL/kg HRW); *p < 0.05; **p < 0.01.